There are more than 250,000 influenza-related deaths each year. Of special concern are the influenza H1N1 and H5N1 strains, which harbor H1 and H5 hemagglutinin (HA) and present potential for future pandemic outbreaks. Current treatments include Tamiflu (oseltamivir) and Relenza (zanamivir), which target the membrane protein neuraminidase (NA). Unfortunately, resistance is increasing in circulating influenza strains. For example, the 2008-2009 H1N1 strain exhibited ~100% resistance against Tamiflu, and thus influenza may be considered as a drug-resistant pathogen. As a consequence, new targets in influenza are urgently needed. HA plays a critical role in influenza entry and thus it i an attractive and novel target for therapeutic intervention. In the first step of influenza entry, A binds to the cellular receptor, sialic acid. Subsequently, the virus enters the cell via receptor-mediated endocytosis. In the endosome, the resulting low pH triggers a large conformational change in HA, which subsequently causes the viral membrane to fuse with the endosomal membrane to allow escape from the endosome. Entry inhibitors could conceivably target either the receptor binding step (binding inhibitors) or the membrane fusion step (fusion inhibitors). In this project we will develop NMR-based screening methods of small molecule libraries to influenza H1 and H5 HA with the long-term goal of developing binding and fusion inhibitors. The Specific Aims are: (1) discovery of chemical probes that bind to influenza H1 and H5 HA in the neutral pH conformation; (2) discovery of chemical probes that bind to influenza H1 and H5 HA in the low pH conformation; (3) identify the site of probe binding by NMR competition experiments. Importantly, the NMR methods could be exploited to identify and improve small molecule therapeutics, characterize probe-envelope interactions in whole virus, and be applied to other viruses such as Ebola, HIV and SARS-CoV.